Antibodies involved in the pathogenesis of chronic idiopathic thrombocytopenic purpura (ITP) react most frequently with platelet glycoprotein (GP) Ib/IX and GPIIb/IIIa. However, uncertainty as to the specificity, frequency, and clinical significance of such antibodies still remains. By using a modified antigen-capture ELISA (MACE), an immunoprecipitation assay, and an immunoblot assay, sera from 60 patients with chronic ITP were analyzed. GP-specific antibodies were found in 50% (30/60) of the patients, with 14 patients having antibodies directed solely to GPIIb/IIIa, 8 holding antibodies specific only for GPIb/IX, and 8 possessing antibodies against both antigens. Serum antibodies were more frequently (p < 0.01) detected in either active and/or non-splenectomized ITP patients. Moreover, in patients displaying antibodies against GPIb/IX, significantly (p < 0.05) lower platelet counts were observed. Using the immunoblot assay, antibodies specific for a 30 kD platelet antigen were detected in 12 of 60 patients. This antigen could not be immunoprecipitated from surface labelled platelet membranes, indicating an intracellular location. We conclude that in chronic ITP, (1) the frequency of anti-GPIIb/IIIa antibodies is close to that of anti-GPIb/IX antibodies, (2) anti-GP antibodies are more likely to be detected in patients with an active disease status and, (3) a 30 kD internal platelet protein is another frequent antigen.